2.1 Cell culture and relative reagents
Different types of human neuroblastoma (NB) cell lines, such as SK-N-AS, SK-N-FI, and SK-N-DZ were performed for the study and obtained from the American Type Culture Collection (Manassas, VA, USA). Cell lines were routinely grown in culture medium (DMEM, Dulbecco’s Modified Eagle’s Medium) with 10% FBS (heat-inactivated fetal bovine serum), GlutaMAX™, nonessential amino acids, and an antibiotic-antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). The cells were maintained at 37 °C in the incubator with 5% CO2 humidified atmosphere. ONC201 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for ATRX were procured from Abcam (Cambridge, MA, USA), β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA), LGR5 from GeneTex (Taipei, Taiwan), c-myc and Ki-67 from Cell Signaling Technology (Danvers, MA, USA).
2.2 Neuroblastoma xenografted animal model
Male NOD/SCID mice, aged four weeks, were procured from the National Laboratory Animal Center (Taipei, Taiwan). The experimental use of animals was cleared by the Institutional Animal Care and Use Committee of Kaohsiung Chang Gung Memorial Hospital, Taiwan (approval number: 2019091805). To establish human neuroblastoma xenografts in the mice, SK-N-AS cells (1×106), SK-N-FI cells (1×107), or SK-N-DZ cells (1×107) were individually injected subcutaneously into the right flank of each mouse. After seven days of implantation, the mice were randomly assigned to different treatment groups. They received either a control treatment (blank culture medium), ONC201 (50 μg/g), 2DG (50 μg/g), or a combined treatment of ONC201 and 2DG (50 μg/g each) administered via intraperitoneal injection once a week for four weeks, following which they were euthanized. Tumor size and weight were recorded as previously described [11]. Tissue samples were fixed by formalin overnight and embedded in paraffin for subsequent immunohistochemical staining.
2.3 Terminal deoxynucleotidyl transferase dUTP nick end-labeling assay
The impact of ONC201 on NB cell viability was quantified using the TUNEL kit (In Situ Cell Death Detection Kit by Roche, Germany) detection. In brief, Cells were fixed with 4% paraformaldehyde for 10 minutes and then incubated with a terminal deoxynucleotidyl transferase and fluorescein-dUTP at 37 °C and subsequently counterstained with DAPI (Southern Biotech, Birmingham, USA) for fluorescence microscopy analysis. The apoptotic index was calculated as the ratio of TUNEL-positive cells to DAPI-positive cells in three randomly selected microscopic fields.
2.4 Western blotting analysis
Post-treatment with 5 μM ONC201 for 48 hours, proteins were extracted from NB cells for analysis. The proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. These membranes were blocked with 5% milk in TBS-T for one hour, then incubated with primary antibodies and HRP-conjugated secondary antibodies for one hour each. Protein complexes were detected by an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Sweden) and recorded on X-ray film.
2.5 Immunohistochemical analysis and scoring
ATRX in NB xenograft tissues and human NB samples were analyzed. Tissue sections were deparaffinized and blocked with 3% hydrogen peroxide, and antigen retrieval was performed using 0.01 M citrate buffer in the microwave at approximately 95°C/10 min. After washing with PBS, sections were incubated with primary antibodies, followed by a peroxidase-conjugated polymer kit (Zymed Laboratories, San Francisco, CA) reaction for 30 minutes. Diaminobenzidine (DAB; Sigma, St. Louis, MO) was used for color development. Sections were counterstained with Gill’s hematoxylin, dehydrated, and mounted. Staining intensity and proportion were scored from 0 (none) to 3 (strong) and 0 (no positive cells) to 4 (81-100% positive cells), respectively. Statistically, these scores are multiplied and summed to obtain a final immunoreactivity score ranging from 0 to 12.
2.6 Generation and characterization of ρ0 cells from neuroblastoma SK-N-AS
The SK-N-AS NB cells were cultured in media containing 50 ng/ml ethidium bromide (EtBr) to remove mitochondrial DNA (mtDNA) for three months and isolate single clones through continuous limiting dilution. The NB ρ0 cells lacking mtDNA were characterized based on mtDNA copy number, expression of mtDNA-encoded proteins, and cellular oxygen consumption.
2.7 Human neuroblastoma tissue biopsies
Tissue samples from 23 patients diagnosed with neuroblastoma, ganglioneuroblastoma, and ganglioneuroma between 2005 and 2022 were obtained from the pathology archives of Chang Gung Memorial Hospital in Kaohsiung, Taiwan. All patients were under 19 years of age at diagnosis. The classification of these cases was based on the International Neuroblastoma Staging System [12], and the histological classification followed the guidelines of the International Neuroblastoma Pathology Committee[13]. The classification system used evaluates the proportion and maturity of neuroblastoma cells and the presence of ganglion cells, classifying tumors into five types: undifferentiated neuroblastoma, poorly differentiated neuroblastoma, differentiated neuroblastoma, ganglioneuroma, and ganglioneuroma.
2.8 Quantification of endothelial cell density after isolectin IB4 staining
Endothelial cell density analysis in the xenografts tissue section was measured by staining with isolectin IB4-biotin conjugates (Molecular Probes). In brief, tissue slides were dewaxed, antigen retrieval was conducted, immunoreactive with antibodies in the sections, and the reaction color was measured using a DAB detection system kit. Five images (200× magnification) were selected from each tumor section to determine endothelial cell density, and the average positive staining area per total pixel was calculated.
2.9 Trypan blue exclusion assay
ONC201 treatments after 24 – 96 hours, cells were stained with 0.4% Trypan Blue (Gibco, Thermo Fisher Scientific Inc.) for 5 minutes and analyzed by Zeiss microscopic (AxioVision, USA) for counted viable cells, which exclude Trypan Blue, appeared clear in the cytoplasm, whereas non-viable cells absorbed the dye and displayed a blue cytoplasm. Counts of viable and non-viable cells were separately recorded to calculate the proportion of cell death.
2.10 Flow cytometry for detection of apoptosis
To evaluate cellular apoptosis, a staining procedure was performed using fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) as per the manufacturer's instructions (BD Pharmingen, BD Biosciences, San Jose, CA, USA). Post-treatment, the cells were suspended in an annexin V binding buffer and stained with annexin V/PI for 15 minutes. A FACS Calibur flow cytometer (BD Biosciences) was used for data collection. Cells positive for annexin V and either negative or positive for PI were identified as undergoing apoptosis.
2.11 Statistical analysis
Statistical assessments were conducted using two-tailed Student's t-tests. Data are presented as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM), with a significance threshold established at p < 0.05. These results are representative of at least three independent experiments.